Hemoglobin-based oxygen carriers (HBOCs) are candidates for use as a blood substitute and resuscitation fluid. We have established that chemical modification used to generate hemoglobins cross-linked at either the alpha or beta chains alter their ability to generate or interact with oxygen free radicals. Exposure of cross-linked hemoglobins to superoxide (O2) generated by the xanthine/xanthine oxidase system causes generation of hydroxyl radicals (.OH) and/or other radicals with similar reactivity. Our results indicate that HBOCs exhibit a significant difference from each other and from unmodified hemoglobins in reactions involving the production of these oxygen free radicals. The relative ability of the ferric derivatives of HBOCs to participate in free radical reactions was monitored by nonenzymatic NADPH oxidation and aniline hydroxylation assays (reactions mediated by reactive oxygen species). Cross-linked hemoglobins again exhibited significant differences in their reactivity with oxygen species such as peroxides and superoxides. In more recent experiments, hydrogen peroxide was produced in a continuous flux, in order to mimic the cellular conditions, by the glucose/glucose oxidase system. Relative to unmodified HbAo, we found that some cross-linked hemoglobins are more susceptible to oxidative modification and the formation of a highly toxic ferryl species. This form of oxidative modification may emerge as a physiologically important event which may lead to a significant contribution to reperfusion injury. A number of in vitro and in vivo studies are planned in order to continue to examine the hemoglobin-mediated radical generation and to determine how these radicals may aggravate "reperfusion injury' in an ischemic animal model. Results of some of these studies were presented at the IV International Symposium on Blood Substitutes, 1991 in Canada. A manuscript describing some of the work has been published in Arch. Biochem. Biophys. Another manuscripts has been submitted for publication in Biochem. Biophys. Res. Comm.